This invention relates to high molecular weight derivatives of vitamin K-dependent polypeptides, and more particularly to dimerized vitamin K-dependent polypeptides and vitamin K-dependent polypeptides that are linked to PEG polymers.
Vitamin K-dependent proteins contain 9 to 13 gamma-carboxyglutamic acid residues (Gla) in their amino terminal 45 residues. The Gla residues are produced by enzymes in the liver that utilize vitamin K to carboxylate the side chains of glutamic acid residues in protein precursors. Vitamin K-dependent proteins are involved in a number of biological processes, of which the most well described is blood coagulation (reviewed in Nelsestuen (2000) Vitam. Horm. 58:355-389). Vitamin K-dependent proteins include protein Z, protein S, prothrombin (factor II), factor X, factor IX, protein C, factor VII, Gas6, and matrix GLA protein. Factors VII, IX, X and II function in procoagulation processes while protein C, protein S and protein Z serve in anticoagulation roles. Gas6 is a growth arrest hormone encoded by growth arrest-specific gene 6 (gas6) and is related to protein S. See, Manfioletti et al. (1993) Mol. Cell. Biol. 13:4976-4985. Matrix GLA protein normally is found in bone and is critical to prevention of calcification of soft tissues in the circulation. Luo et al. (1997) Nature 386:78-81.
The regulation of blood coagulation is a process that presents a number of leading health problems, including both the failure to form blood clots as well as thrombosis, the formation of unwanted blood clots. Agents that prevent unwanted clots are used in many situations and a variety of agents are available. Unfortunately, most current therapies have undesirable side effects. Orally administered anticoagulants such as Warfarin act by inhibiting the action of vitamin K in the liver, thereby preventing complete carboxylation of glutamic acid residues in the vitamin K-dependent proteins, resulting in a lowered concentration of active proteins in the circulatory system and reduced ability to form clots. Warfarin therapy is complicated by the competitive nature of the drug with its target. Fluctuations of dietary vitamin K can result in an over-dose or under-dose of Warfarin. Fluctuations in coagulation activity are an undesirable outcome of this therapy.
Injected substances such as heparin, including low molecular weight heparin, also are commonly used anticoagulants. Again, these compounds are subject to overdose and must be carefully monitored.
A newer category of anticoagulants includes active-site modified vitamin K-dependent clotting factors such as factor VIIa and IXa. The active sites are blocked by serine protease inhibitors such as chloromethylketone derivatives of amino acids or short peptides. The active site-modified proteins retain the ability to form complexes with their respective cofactors, but are inactive, thereby producing no enzyme activity and preventing complexing of the cofactor with the respective active enzymes. Thus, active-site modified Factor VIIa, denoted factor VIIai, still binds tissue factor, but does not have enzyme activity. Active site-modified proteins appear to have very beneficial anti-coagulant properties with few undesirable side affects. For example, factor VIIai has been shown to lower platelet deposition at the site of surgery, an important indicator of anti-coagulation action. While this can also be accomplished by heparin or other anticoagulants, factor VIIai was unique in that its administration was not accompanied by increased bleeding time or blood loss. See, Harker et al. (1997) Thromb. Haemost. 78:736-741. A similar outcome was reported when factor IXai was administered during surgery. See, Spanier et al. (1998) J. Thorac. Cardiovasc. Surg. 115(5):1179-88. In short, these proteins appear to offer the benefits of anticoagulation therapy without the adverse side effects of other anticoagulants. Active site modified factor Xa is another possible anticoagulant in this group. Its cofactor protein is factor Va. Active site modified activated protein C (APC) may also form an effective inhibitor of coagulation. See, Sorensen et al. (1997) J. Biol. Chem. 272:11863-11868. Active site modified APC binds to factor Va and prevents factor Xa from binding.
A major inhibition to the use of active site-modified vitamin K-dependent clotting factors is cost. Biosynthesis of vitamin K-dependent proteins is dependent on an intact glutamic acid carboxylation system, which is present in a small number of animal cell types. Overproduction of these proteins is severely limited by this enzyme system. Furthermore, the effective dose of these proteins is high. A common dosage is 1000 xcexcg of VlIIai/kg body weight. See, Harker et al. 1997 supra. Current cost (April of 2000) of recombinant factor VIIa is about $0.80 per xcexcg, which severely limits use.
A second problem for several of these proteins is a short lifetime in the circulation system. The situation for factor VIIa illustrates this problem. Factor VII and VIIa have circulation half-times of about 2-4 hours in the human. That is, within 2-4 hours, half of the protein is taken up by other tissues of the body. When factor VIIa is used as a procoagulant to treat certain forms of hemophilia, the standard protocol is to inject VIIa every two hours and at high dosages (45 to 90 xcexcg/kg body weight). See, Hedner et al. (1993) Transfus. Med. Rev. 7:78-83. Thus, use of these proteins as procoagulants or anticoagulants (in the case of factor VIIai) requires that the proteins be administered at frequent intervals and at high dosages.
The invention is based, in part, on modifications to vitamin K-dependent polypeptides that increase their circulation half-life and in some embodiments, their activity. Both outcomes reduce the amount of protein needed to treat clotting disorders as well as decrease the frequency of administration. As a result, costs associated with treating patients can be reduced, allowing the therapies to be made more widely available to individuals in need of pro- or anti-coagulation therapies.
In one aspect, the invention features an isolated vitamin K-dependent polypeptide linked (e.g., directly or indirectly) to a polyethylene glycol (PEG) polymer. The polypeptide can be selected from the group consisting of factor VII, factor IX, factor X, factor II, protein C, protein S, gas6, and bone matrix Gla protein or can be a protease selected from the group consisting of factor VIIa, factor IXa, factor Xa, factor IIa, and activated protein C. Factors VIIa, IXa, and Xa are particularly useful proteases. The protease can be further linked to an active-site inhibition reagent such as a chloromethylketone derivatized amino acid or peptide. In some embodiments, the PEG polymer is linked to the protease via the active-site inhibition reagent.
The invention also features an active-site inhibition reagent linked to a PEG polymer. The reagent can be a chloromethylketone derivatized amino acid or peptide or a phosphohalide derivative.
In another aspect, the invention features an anticoagulant agent that includes two polypeptide monomers, wherein at least one of the polypeptide monomers is a vitamin K-dependent polypeptide, and wherein the polypeptide monomers are covalently linked. The polypeptide monomers can be covalently linked via a bi-functional active-site inhibition reagent. The two polypeptide monomers can be the same or different polypeptides. In some embodiments, each of the two polypeptide monomers is a vitamin K-dependent polypeptide, such as a factor VIIa polypeptide, a factor Xa polypeptide, or a factor IXa polypeptide. The bi-functional active-site inhibition reagent can be linked to a PEG polymer. At least one of the polypeptide monomers also can be directly linked to a PEG polymer.
The invention also features a bi-functional active-site inhibition reagent that includes two covalently linked active-site inhibitors. At least one of the active-site inhibitors can be linked to a PEG polymer.
A method of directly linking a vitamin K-dependent polypeptide to a PEG polymer also is featured. The method includes incubating the PEG polymer with the vitamin K-dependent polypeptide for a time sufficient to link the PEG polymer to the vitamin K-dependent polypeptide, wherein the PEG polymer is reactive with amino groups or carbohydrate groups on the vitamin K-dependent polypeptide.
In yet another aspect, the invention features a method of indirectly linking a vitamin K-dependent polypeptide to a PEG polymer. The method includes providing a PEG-modified, active-site inhibition reagent, wherein the PEG polymer is reactive with amino groups on the active-site inhibition reagent; and incubating the PEG-modified, active-site inhibition reagent with the vitamin K-dependent polypeptide for a time sufficient to link the PEG modified, active-site inhibition reagent to the vitamin K-dependent polypeptide.
A method of making an anticoagulant agent also is featured. The method includes incubating a bi-functional active-site inhibition reagent and at least one vitamin K-dependent polypeptide in the presence of phospholipid for a time sufficient to link the bi-functional active-site inhibition reagent and the vitamin K-dependent polypeptide.
In another aspect, the invention features a pharmaceutical composition that includes an isolated vitamin K-dependent polypeptide linked to a PEG polymer and a pharmaceutically acceptable carrier. The polypeptide and PEG polymer can be indirectly linked. The polypeptide can be a protease selected from the group consisting of factor VIIa, factor IXa, factor Xa, factor IIa, and activated protein C. Factors VIIa, IXa, and Xa are particularly useful proteases. The protease can be further linked to an active-site inhibition reagent such as a chloromethylketone derivatized amino acid or peptide. The PEG polymer can be linked to the protease via the active-site inhibition reagent.
The invention also features a pharmaceutical composition that includes an anticoagulant agent and a pharmaceutically acceptable carrier, wherein the anticoagulant agent includes two polypeptide monomers, wherein at least one of the polypeptide monomers is a vitamin K-dependent polypeptide, and wherein the polypeptide monomers are covalently linked. The polypeptide monomers can be covalently linked via a bi-functional active-site inhibition reagent.
In yet another aspect, the invention features a method for evaluating dosage of factor VIIa. The method includes obtaining a biological sample from a patient undergoing factor VIIa therapy; and monitoring clotting time of the biological sample in a device, wherein the device comprises an activator of the contact phase of coagulation and is lacking added phospholipid, wherein a sufficient decrease in clotting time compared to a control sample from the patient before the factor VIIa therapy indicates that an appropriate dosage of factor VIIa has been administered. Factor VIIa therapy can include administering to the patient factor VIIa linked to a PEG polymer.
The invention also features a method for managing anticoagulation therapy in a patient. The method includes administering an acute phase anticoagulant to the patient during the acute phase of coagulation; and administering an active-site inhibited factor VIIa polypeptide to the patient during the chronic phase of coagulation. The active-site inhibited factor VIIa polypeptide can be linked to a PEG polymer, as described above. The acute phase anticoagulant can be active-site modified factor IXa or active-site modified Xa.
Pharmaceutical compositions that include an active-site inhibited factor VIIa polypeptide and an acute phase anticoagulant also are featured. The active-site inhibited factor VIIa polypeptide can be linked to a PEG polymer, as described above. The PEG polymer can be linked to the active-site inhibited factor VIIa polypeptide via an active-site inhibition reagent.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.